Cellular proliferation in pilocytic and diffuse astrocytomas

Using quantitative image analysis, we evaluated the MIB-1 labeling index (LI) in a large population of pilocytic (n = 131) and diffuse astrocytomas (n = 140), explored its significance as a prognostic predictor of survival, and compared it to other commonly accepted predictors, including grade and its histologic determinants, atypia, mitoses, endothelial proliferation, and necrosis. Diffuse astrocytomas were graded according to the St Anne-Mayo scheme and included 45 grade 2, 50 grade 3, and 45 grade 4 astrocytomas. In pilocytic astrocytomas, mean, median, and range of MIB-1 LIs were 1.1, 0.9, and 0-3.9%, respectively. In diffuse astrocytomas, these values were 2.3, 2, and 0-7.6% in grade 2; 6, 4.4, and 0.1-25.7% in grade 3; 9.1, 6, and 0.3-36% in grade 4. There was a significant difference in the distribution of MIB-1 LIs between pilocytic and diffuse grade 2 astrocytomas (p < 0.001), between grade 2 and grade 3 (p < 0.001), and between tumors of grade 3 and 4 (p = 0.014). Among pilocytic astrocytomas there was no association between survival and MIB-1 LI or any histologic parameter. In diffuse astrocytomas, MIB-1 LI was significantly correlated with grade as well as with mitotic activity (<0.001) and survival. While in diffuse astrocytomas of all grades, necrosis was the strongest factor associated with survival, in tumors of grades 2 and 3 the MIB-1 LI preceded other histologic parameters and, on multivariate analysis, remained the only feature predictive of survival. Grade 3 astrocytomas with a single "solitary" mitosis had a significantly lower MIB-I LI than did grade 3 tumors with >1 mitosis and, compared to the latter, had a significantly longer survival (p = 0.013), one not significantly different from patients with grade 2 astrocytomas. These findings suggest that the cutoff point between grade 2 and 3 in the St. Anne-Mayo scheme may not be optimal and may need to be revised.     

The expression of cell cycle regulatory proteins in oligodendroglial tumors

Oligodendroglial tumors are mainly classified histologically into 2 types of tumors--oligodendrogliomas and oligoastrocytomas--whether the tumor includes astrocytic component or not, and these are, respectively, divided histologically into 2 different malignant groups, low-grade (WHO grade II) and high-grade or anaplastic (WHO grade III). In this study, we investigated the expression of the cell cycle-related proteins and analyzed the relationship between the labeling index (LI: the percentage of positive nuclei) of these proteins and 2 histopathological phenotypes, grade and prognosis. Forty-four specimens were examined, including 11 oligodendorogliomas (0), 5 oligoastrocytomas (OA), 18 anaplastic oligodendrogliomas (aO) and 10 anaplastic oligoastrocytomas (aOA), according to the WHO classification. Of these, 19 specimens were obtained from recurrent tumors of 8 cases. The mean LI of all the investigated proteins between O and OA, and aO and aOA showed no significant differences. The mean MIB-1 LI, pRb LI, p53 LI and p14 LI of GIII were significantly higher than GII, while the mean p27 LI of GIII was significantly lower than GII. In the recurrent cases, we noted correlations between the disease-free period and MIB-1 LI (inverse), p27 LI and p14 (inverse). Significant correlations were noted between MIB-1 LI and pRb LI, MIB-1 LI and cycD1 LI, MIB-1 LI and p27 LI (inverse), pRb LI and cycD1 LI, cycD1 LI and cdk4 LI and pRb LI and p27 LI (inverse) in the pRb/cycD1-cdk4/p27 pathway, and between MIB-1 LI and p53 LI, MIB-1 LI and p14 LI, p53 LI and p14 LI, p53 LI and MDM2 LI and MDM2 LI and p14 LI in the p53/MDM2/p14 pathway. In conclusion, both pRb/cycD1-cdk4/p27 and p53/N4DM2/p14 pathways correlate with malignant progression, and MIB-1 LI, pRb LI, p27 LI, p53 LI and p14 LI reflect the histopathological malignancy of oligodendroglial tumors. MIB-1 LI, pRb LI and p27 LI are especially useful as indicators of malignancy of oligodendroglial tumors.     

p53 protein alterations in adult astrocytic tumors and oligodendrogliomas

BACKGROUND: p53 is a tumor suppressor gene implicated in the genesis of a variety of malignancies including brain tumors. Overexpression of the p53 protein is often used as a surrogate indicator of alterations in the p53 gene. AIMS: In this study, data is presented on p53 protein expression in adult cases (>15 years of age) of astrocytic (n=152) and oligodendroglial (n=28) tumors of all grades. Of the astrocytic tumors, 86% were supratentorial in location while remaining 14% were located infratentorially - 8 in the the cerebellum and 13 in the brainstem. All the oligodendrogliomas were supratentorial. MATERIALS AND METHODS: p53 protein expression was evaluated on formalin-fixed paraffin-embedded sections using streptavidin biotin immunoperoxidase technique after high temperature antigen retrieval. RESULTS: Overall 52% of supratentorial astrocytic tumors showed p53 immunopositivity with no correlation to the histological grade. Thus, 58.8% of diffuse astrocytomas (WHO Grade II), 53.8% of anaplastic astrocytomas (WHO Grade III) and 50% of glioblastomas (WHO Grade IV) were p53 protein positive. In contrast, all the infratentorial tumors were p53 negative except for one brainstem glioblastoma. Similarly, pilocytic astrocytomas were uniformly p53 negative irrespective of the location. Among oligodendroglial tumors, the overall frequency of p53 immunopositivity was lower (only 28%), though a trend of positive correlation with the tumor grade was noted - 25% in Grade II and 31.5% in grade III (anaplastic oligodendroglioma). Interestingly, p53 labeling index (p53 LI) did not correlate with the histopathological grade in both astrocytic and oligodendroglial tumors. CONCLUSIONS: Thus, this study gives an insight into the genetic and hence biological heterogeneity of gliomas, not only between astrocytic tumors vs. oligodendrogliomas but also within astrocytic tumors with regard to their grade and location. With p53 gene therapy trials in progress, this will possibly have future therapeutic implications.     

High cyclin E/low p27Kip1 expression is associated with poor prognosis in astrocytomas

Cyclin E and p27Kip1 are co-regulators of the G1- to S-phase transition and closely related to tumor behavior. The purpose of this study was to examine expression of cyclin E and p27Kip1 in astrocytomas and to evaluate the relationships between expression of these cell-cycle regulators and prognosis of patients with astrocytoma. Tissue samples from 130 astrocytomas (WHO grade 1 n = 5, grade 2 n = 23, grade 3 n = 64, grade 4 n = 38) were examined immunohistochemically for cyclin E and p27Kip1 expression. Patient charts were reviewed for clinical presentation, and survival was followed. The cyclin E labeling index (LI) tended to increase with tumor grade (Kruskal-Wallis, P = 0.0104). For patients with primary astrocytomas, the 50% survival times for the low cyclin E LI (< 5%) group and the high cyclin E LI (> or = 5%) group were 53.7 months and 19.8 months. In combined analysis of cyclin E and p27Kip1 expression, the low cyclin E/high p27Kip1 LI (> or = 50%) group had the best survival (50% survival time: 103.2 months), the low cyclin E/low p27Kip1 LI (> or = 50%) and the high cyclin E/high p27Kip1 LI groups moderate survival (24.1 and 27.5 months), and the high cyclin E/low p27Kip1 LI group the worst survival (13.1 months). Multivariate analysis identified the combined factor, high cyclin E/low p27Kip1, as a novel independent prognostic factor for survival time (P = 0.0037, relative risk = 2.4). This study suggested that combined analysis of cyclin E and p27Kip1 expression was considered to be potentially useful in predicting the prognosis of patients with astrocytoma.     

Correlation between histological grade and MIB-1 and p53 immunoreactivity in meningiomas

OBJECTIVE: Meningiomas for the most part are slow-growing benign tumors, but complete removal can be difficult and recurrence is an issue. The aim of this study was to re-evaluate tumors diagnosed as meningioma previously in our hospital, according to the latest World Health Organization classification. We also examined the relationships among parameters such as brain invasion, histological grade and Ki-67 and p53 expression in these tumors. MATERIALS AND METHODS: Meningioma biopsy specimens numbering 60 (48 grade I, 11 grade II, and 1 grade III tumors) were examined immunohistochemically using monoclonal antibodies for Ki-67 (MIB-1) and p53 protein. The MIB-1 labeling index (LI) for each tumor was calculated as a percentage based on the number of stained cells per total cells counted. The level of p53 expression in each sample was semiquantatively evaluated as < 1%, 1 - 10%, 10 - 70%, or > 70%. Any value > 1% was accepted as presence of p53 expression. RESULTS: Of the 60 meningiomas, 7 (11.7%) exhibited brain invasion. The mean MIB-1 LI values for the grade I and grade II tumors were 1.1% and 2.3%, respectively. The corresponding levels of p53 protein expression in these groups were 54.1% and 72.7%. The MIB-1 LI and the level of p53 expression in the one grade III meningioma were 6.7% and 10 - 70%, respectively. Histological grade was significantly correlated with MIB-1 LI and with p53 expression (p < 0.01 for both). Brain invasion was not correlated with histological grade, MIB-1 LI, or p53 expression. CONCLUSION: The results indicate that MIB-1 LI and p53 protein expression are good indicators of histological grade in meningioma and may be particularly valuable for distinguishing borderline atypical meningiomas. The number of cases was limited, but the findings also suggest that brain invasion is a prognostic parameter independent of grade, MIB-1 LI and p53 expression.     

自噬相关基因Beclin1在星形细胞瘤中的表达及意义

目的探讨自噬相关基因Beclin1在星形细胞瘤中的表达,及与肿瘤病理和临床表现的相关性与意义。方法采用免疫组化和免疫印迹法检测62例不同级别的星形细胞瘤Beclin1的表达情况,并结合临床表现和病理结果进行相关性分析。结果免疫组化显示星形细胞瘤中Beclin1蛋白表达随肿瘤级别升高而降低。免疫印迹法显示高级别（Ⅲ～Ⅳ级）星形细胞瘤Beclin1平均光密度比值低于低级别（Ⅰ～Ⅱ级）肿瘤（P=0.036）。生存期大于36个月者Beclin1平均光密度比值高于生存期小于36个月者（P=0.041）。结论Beclin1在高级别星形细胞瘤中表达下降,Beclin1检测可作为判断生存期和临床预后的辅助指标。     

Gliomas of the optic nerve: histological, immunohistochemical (MIB-1 and p53), and MRI analysis

Gliomas of the optic nerve, although typically of pilocytic (WHO grade I) histology, can present within the spectrum of astrocytic neoplasia including glioblastoma (WHO grade IV). In certain cases, histologic features alone make the distinction between pilocytic and diffuse astrocytomas difficult. We reviewed 22 cases of optic nerve gliomas, 19 of which were pilocytic astrocytomas (PA), and 3 of which were diffuse, non-pilocytic astrocytomas. The cases were evaluated for their clinical course, radiographic appearance, histologic grade, and proliferation indices as detected by MIB-1 (Ki-67) and p53 antibodies. Of the 19 PA, 14 showed no tumor growth by magnetic resonance imaging, and had Ki-67 and p53 labeling indices (LI) of < 1%. The other 5 PA exhibited aggressive behavior manifest by marked diffuse infiltrative tumor growth causing death in 2 patients, 1 of whom was diagnosed with neurofibromatosis type 1 (immunoperoxidase and radiographs not available), and marked local growth with an average time to growth of 39.3 months, a Ki-67 LI of 2–3%, and a p53 LI of < 1% in three others. Three of the five aggressive PA histologically demonstrated a finely reticulated pattern, a pattern that appears as an exaggeration or expansion of the normal neuroglia of the optic nerve, and may simulate a diffuse low-grade astrocytoma. Two demonstrated the coarsely reticulated pattern, with the biphasic and microcystic pattern typical of PA. Three diffuse astrocytomas (2 anaplastic astrocytomas and 1 glioblastoma) originated clinically and radiographically from the optic nerve, and revealed a Ki-67 LI of 2–12%, a p53 LI of 2–8%, and an average time to growth of 8 months. We conclude that the majority of PA of the optic nerve are non-aggressive, stabilize radiographically, and have Ki-67 and p53 LI < 1%. However, a subpopulation of PA has a propensity for aggressive behavior, and are identified by a Ki-67 LI of 2–3% and a p53 LI of < 1%. Diffuse astrocytomas have both Ki-67 and p53 LI > 2%. Thus, in cases of aggressive optic nerve tumors in which the histologic review of biopsy material cannot confidently confirm the diagnosis of pilocytic or diffuse fibrillary glioma, a p53 LI of > 1% appears to favor the diagnosis of diffuse astrocytoma. Received: 18 May 1999 / Revised: 10 August 1999 / Accepted: 18 August 1999     

Diagnostic validity of the Ki-67 labeling index using the MIB-1 monoclonal antibody in the grading of meningiomas

BACKGROUND: About 10% of meningiomas behave aggressively and are graded atypical or malignant with important therapeutic and prognostic implications. Routine histological parameters are inconsistent in the assessment of their aggressive behavior. AIMS: The aim of this study was to find a threshold level of the MIB-1 labeling index (MIB-1 LI) with the highest diagnostic validity in predicting histological atypia in a meningioma. SETTING AND DESIGN: This was a retrospective study of all atypical and malignant meningiomas diagnosed at our center between January 1995 and June 2000 and which were identified from the General Pathology Registry. MATERIAL AND METHODS: These meningiomas were assessed histologically with respect to the individual criteria of atypia. They were categorized according to the WHO 2000 classification as benign, atypical and anaplastic meningiomas, WHO Grades I, II and III respectively and by immunohistochemical analysis using the MIB-1 monoclonal antibody. STATISTICAL ANALYSIS: The diagnostically useful cut-off level for the prediction of atypia was estimated by calculating the sensitivity and specificity of the MIB-1 LI at various levels and a receiver operated characteristic (ROC) analysis was performed. The correlation between the individual histological parameters was studied and the MIB-1 LI was obtained using Fisher's exact test. RESULTS: Of the 40 meningiomas studied 21 were benign, 16 atypical and 3 anaplastic. Atypical tumors had a higher MIB-1 LI than benign tumors, with diagnostic validity highest at a threshold of 7%, with a sensitivity of 0.86 and a specificity of 0.93, giving a likelihood ratio of 17. The MIB-1 LI correlated well with mitotic activity and the other individual criteria in the WHO 2000 definition of atypia in a meningioma. MIB-1 LI did not, however, correlate well with brain invasion. CONCLUSION: The MIB-1 LI has the highest validity in the diagnosis of atypia in meningiomas at a threshold level of 7%. The MIB-1 LI used in conjunction with histological features can help in making a recommendation regarding potentially aggressive behavior in meningiomas.     

Prognostic implication of histopathological, immunohistochemical and clinical features of oligodendrogliomas: a study of 89 cases

Histopathological, immunohistochemical and clinical parameters were correlated with survival in 89 cases of oligodendroglioma (65 patients with grade II and 24 patients with grade III of the WHO classification). Median survival time and 5-year survival rate were 3.5 years and 76% for patients with oligodendroglioma grade II and 0.875 years and 23% for patients with oligodendroglioma grade III. The tumor biopsy specimens were immunohistochemically analyzed for Ki 67 (MIB-1), vimentin, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE) and synaptophysin. MIB-1 nuclear labeling index ranged from 0.0% to 33.4%; vimentin-immunoreactive tumor cells were found in 25 cases. MIB-1 nuclear labeling index and vimentin immunoreaction showed a significant statistical correlation to the 5-year survival rate of the patients. Tumors with vimentin expression (n = 25) and/ or high MIB-1 labeling index (n = 26) had a poorer prognosis than tumors lacking vimentin expression (n = 57) and/or displaying a low MIB-1 labeling index (n = 56). The expression of immunoreactivity for GFAP (n = 53), NSE (n = 23) and synaptophysin (n = 15) appeared to be of no prognostic relevance. Patients with gross total tumor resection (n = 47) had a median survival time and 5-year survival rate of 3.3 years and 84% compared to 1.2 years and 42% for patients with subtotal resection (n = 41). The comparison between patients who underwent surgery alone (n = 53) and those who had surgery plus postoperative radiation therapy showed no significant survival benefit from postoperative radiation therapy. In conclusion, tumor grade, MIB-1 labeling index, expression of vimentin and the extent of surgery are shown to be of prognostic relevance for patients with oligodendroglioma. Received: 29 July 1997 /Revised: 14 October 1997, 4 December 1997 / Accepted: 14 December 1997     

Evaluation of p53 protein expression and proliferative potential based on the MIB-1 positive index in astrocytic gliomas

Fifty-seven patients with infiltrative astrocytic gliomas, comprising 29 cases of glioblastoma, 13 cases of anaplastic astrocytoma and 15 cases of low-grade astrocytoma, were examined. In 12 cases of astrocytic glioma, the p53 gene mutation was investigated by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). p53 overexpression was observed in 20 (35%) of the 57 patients with astrocytic gliomas. The occurrence of p53 overexpression was apparently correlated with the histological grading of the astrocytic gliomas. Of the 20 patients with glioblastoma who were less than 60 years old (mean age ± SD, 39.2 ± 15.0 years), 50% demonstrated p53 overexpression, whereas only 2 (22%) patients with glioblastoma who were more than 60 years old (mean age ± SD, 65.1 ± 5.8 years) had p53 overexpression. A substantial difference in survival associated with glioblastoma was noted between the p53-positive group (mean survival 28.9 months) and the p53-negative group (mean survival 11.3 months) based on Kaplan-Meier survival curves (P= 0.01). Even among patients with glioblastoma who were less than 60 years old, a better survival rate was recognized in those with p53 overexpression than in those without p53 (P= 0.03). The MIB-1 indices tended to increase with tumor malignancy, and a poor survival time was significantly associated with elevated values for the MIB-1 index. A substantial difference in survival between the two groups of patients who had MIB-1 indices of above 5% and below 5% was evident from the Kaplan-Meier survival curves (P= 0.04). The group that had no p53, and MIB-1 indices of above 5%, was found to show the malignant gliomas most frequently. Although most of the gliomas with p53 overexpression demonstrated MIB-1 indices of above 5%, there was no significant correlation between p53 protein expression and the MIB-1 index. Of the 12 patients with astrocytic glioma who were examined by PCR-SSCP, 3 (one glioblastoma and 2 anaplastic astrocytomas) revealed p53 gene mutation correlated with p53 (DO-7) overexpression.     

Distribution of nuclear size and internuclear distance are important criteria for grading astrocytomas

AIM: The differentiation between low-grade astrocytomas and anaplastic astrocytomas is susceptible to considerable inter-observer variability. In order to contribute to a better standardization of astrocytoma-grading based on quantitative data, the present study focuses on two important aspects not being considered in previous morphometric studies: elaboration of a decision flow chart for tumor grading based on morphometric parameters and appropriate cut-off-values, easily performed using low-cost equipment such as measuring oculars; investigation of the distribution (histograms) of parameters describing nuclear size and internuclear distance, which had been represented in previous studies by their mean and standard deviation only. MATERIAL AND METHODS: At least 300 tumor cell nuclei per case were investigated in paraffin sections from surgical specimen of 75 patients with astrocytomas WHO grade II (n = 23) and anaplastic astrocytomas WHO grade III (n = 52) by means of a digital image analysis system. RESULTS: The morphometric data showed significant differences between both groups of tumors. According to multivariate analysis, the best contribution to tumor grading was achieved by means of parameters concerning the distribution of values for nuclear diameters and internuclear distances. A decision tree was constructed using a knowledge based algorithm, which provided astrocytoma grading based on the distribution of values for nuclear diameter, as well as the numerical nuclear density and proliferation index. Measurements using a measuring ocular took an acceptable amount of time (1.5 hour per case) and showed good reproducibility when compared with measurement by means of digital image analysis. CONCLUSION: The study demonstrates that a morphometric examination of tumor cell nuclei in paraffin sections supports the clinically important differential diagnosis between low-grade and high-grade astrocytomas. The method for classification and the data published in the present study constitute a good basis for a standardized and reproducible grading procedure for astrocytomas, which can be performed in any histologic laboratory even without a digital image analysis system.     

微管相关蛋白LC3B-Ⅱ和自噬基因Beclin1在星形细胞肿瘤中的表达及其意义

目的 探讨微管相关蛋白1轻链3B(MAP1-LC3B)和自噬相关基因Beclin1在星形细胞肿瘤中的表达及其与星形细胞肿瘤病理和临床表现的相关性及意义.方法 选择上海长征医院神经外科2006年1月至2006年12月住院手术治疗的初发性星形细胞肿瘤患者62例,其中Ⅰ级(毛细胞性星形细胞瘤)4例,Ⅱ级(星形细胞瘤)23例,Ⅲ级(间变性星形细胞瘤)12例,Ⅳ级(胶母细胞瘤)23例.免疫组织化学染色检测肿瘤标本中Beclin 1的表达,免疫印迹法检测Beclin 1和MAP1-LC3B的表达并统计Beclin 1、MAP1-LC3B的表达与星形细胞肿瘤临床病理表现的关系.结果免疫组化显示Ⅰ～Ⅳ级星形细胞肿瘤中Beclin 1的表达随肿瘤级别的升高而降低,差异有统计学意义(P＜0.05).免疫印迹检测结果显示不同病理分级、生存期患者Beclin 1的表达差异有统计学意义(P＜0.05),不同病理分级患者MAP1-LC3B-Ⅱ表达的差异有统计学意义(P＜0.05);相关性分析显示MAP1-LC3B-Ⅱ、Beclin 1在星形细胞肿瘤中的表达与病理分级呈负相关关系(r=-0.334,P=0.007;r=-0.448,P=0.000),MAP1-LC3B-Ⅱ、Beclin 1在星形细胞肿瘤中的表达与生存时间呈正相关关系(r=0.285,P=0.027;r=0.359,P=0.005).MAP1-LC3B-Ⅱ和Beclin 1的表达之间也呈正相关关系(r=0.272,P=0.035).结论 MAP1-LC3B-Ⅱ和Beclin 1在胶母细胞瘤中的表达下调,自噬活性的改变可能与星形细胞肿瘤的发生、发展相关.

Abstract：

Objective To investigate the expressions of microtubule -associated protein 1 (MAP1) light chain 3B (LC3B) and autophagy-related gene Beclin1 in astrocytic tumors, and explore their correlations with the pathological features and clinical manifestations of astrocytic tumors to further reveal their roles in tumorigenesis and development of astrocytic tumors. Methods Sixty-two specimens with different-grade astrocytic tumors, including 4 with grade Ⅰ (pilocytic astrocytoma), 23 with grade Ⅱ (astrocytoma), 12 with grade Ⅲ (anaplastic astrocytoma) and 23 with grade Ⅳ (glioblastoma multiforme), were selected in our study. Immunohistochemistry was employed to detect the expression of Beclin1; the expressions of MAP 1-LC3B and Beclin1 were detected by Western blotting.The correlations between expressions of MAP 1-LC3B and Beclin 1 and both the pathological features and clinical manifestations of astrocytic tumors were analyzed. Results Immunohistochemistry showed decreased Beclin1 expression in the astrocytic tumors following the increase of tumor grades (P＜0.05).Western blotting indicated that the expressions of Beclin1 in tumors with different grades and these patients with different life cycles were significantly different (P＜0.05) and the average optical density ratio of Beclin1 in high-grade astrocytic tumors (grade Ⅲ/Ⅳ) was obviously lower than that in low-grade astrocytic tumors (grade Ⅰ/Ⅱ, P＜0.05). The expressions of LC3B-Ⅰ showed significant differences in different-grade astrocytic tumors, and the expression of LC3B-Ⅰ of grade Ⅳ tumor was statistically lower than that of grade Ⅰ, Ⅱ and Ⅲ tumors(P＜0.05). The expressions of LC3B-Ⅱ and Beclin 1 were negatively correlated to the pathological grade of the tumors (r=-0.334, P=0.007; r=-0.448, P=0.000), but positively correlated to the survival time(r=0.285, P=0.027; r=0.359, P=0.005). The expressions of LC3B-Ⅱ and Beclin 1 had a positive correlation (r=0.272, P=0.035). Conclusion Expressions of LC3B-Ⅱ and Beclin1 are down-regulated in glioblastoma multiforme; the decrease of autophagic capacity may relate to the tumorigenesis and development of astrocytic tumors.     

Biologic parameters that correlate with the prognosis of human gliomas

Much clinical and biologic data have been processed in the search for useful objective parameters to predict brain tumor behavior. Seventy cases of astrocytic glioma collected by a single clinical team were studied using a full complement of clinical procedures: follow up (7 years), histologic analysis, DNA content estimation, and cell kinetics by flow cytometry. Proliferating cell nuclear antigen (PCNA) was determined by immunocytochemical‐coupling flow cytometry (PFC) and also by counting under light microscopy (PIHC). A statistical evaluation was carried out to establish the usefulness of several parameters for glioma prognosis. The cases were histologically classified as 14 low‐grade astrocytomas, 20 anaplastic astrocytomas, and 36 glioblastomas multiforme. The survival curve showed significant differences between his‐tologic groups. Diploid populations were more frequent in low‐grade astrocytomas, and aneuploid tumors often had increased S‐phase and proliferative fractions. The PCNA‐labeled index (PCNA‐LI) increased with malignancy and correlated with histologic grading (P = 0.01). The PCNA‐LI and age segregated low‐ from high‐grade astrocytomas (including anaplastic astrocytoma and glioblastoma multiforme), but none of the variables considered differentiated anaplastic astrocytoma from glioblastoma multiforme. The Cox regression test displayed significant values for age, histologic diagnosis, and PCNA determinations when considered in tandem. Discriminant analysis obtained a function integrating age and specifically PIHC‐LI to help in the prognosis of doubtful cases. The results emphasize the importance of parameters integrating different variables in an attempt to provide an accurate prognosis, the most significant being age, histopathologic diagnosis, and the proliferative fraction determined by PCNA.     

Cyclin D1 expression in gliomas

Cyclin D1 (cycD1) expression was defined immunohistochemically using monoclonal antibody DCS-6 and polyclonal antiserum H-295 in 50 glioma biopsies. The number of positive nuclei was higher for H-295 than for DCS-6, with a ratio of 3:1. The labelling index (LI) was compared to the grade of histological malignancy and to Ki-67 MIB-1 LI. The LI for cycD1 increased with histological malignancy, in parallel with the increase in MIB-1 LI. In most tumours, the maximum LI for cycD1 and MIB-1 were found in the same areas. The mean MIB-1 LI: mean cycD1 LI ratio does not vary in the three grades of astrocytic tumours. However, in this study the correlation between the two LIs was not statistically significant. Staining for cycD1 antigen does not necessarily imply that the gene is overexpressed since other molecular mechanisms can also be responsible for cell cycle deregulation. In invasive areas, the cycD1 LI is frequently higher than in solid tumour, either because more tumour cells are positive or because reactive astrocytes and activated microglia express cycD1. The relative contribution of neoplastic and reactive cells remains to be defined. Received: 23 May 1997 / Revised: 21 July 1997 / Accepted: 20 August 1997     

Expression of retinoblastoma gene product and p21 (WAF1/Cip 1) protein in gliomas: correlations with proliferation markers, p53 expression and survival

Using immunohistochemistry we evaluated the expression of two negative regulators of the cell cycle, the retinoblastoma gene product (pRb) and the WAF1/Cip1 gene product (p21), in consecutive paraffin sections from 54 gliomas (49 astrocytomas and 5 oligodendrogliomas) and related it to clinicopathological parameters, proliferative fraction, p53 expression and survival. Survival analysis was restricted to the group of diffuse astrocytomas (48 patients). pRb expression did not correlate with histological type, grade or p53 expression, while a moderately strong correlation existed between pRb expression and the percentages of proliferating cell nuclear antigen (PCNA) and MIB-1-positive cells. In 30% of cases we observed diminished pRb expression (i.e., a low pRb/Ki-67 ratio), irrespective of grade or histological type. p21 protein was elevated in 50% of cases, especially within the higher grades. The percentage of p21-positive cells was not related to histological type or grade but correlated loosely with PCNA and pRb expression. A p53-negative/p21-negative phenotype was characteristic of oligodendrogliomas and low-grade astrocytomas, whereas the p53-positive/p21-positive, p53-positive/p21-negative and p53-negative/p21-positive phenotypes were almost equally distributed among high-grade tumors. In survival analysis (either univariate or multivariate) diminished pRb expression was not a statistically significant prognostic indicator. In contrast, p21 expression emerged as an important indicator of shortened disease-free survival, in both univariate and multivariate analyses. Moreover, the double-positive p53/p21 phenotype tended to be associated with a shorter overall survival. Our results suggest that Rb gene deregulation does not significantly affect prognosis but p21 expression may play an important role in disease-free survival of astrocytoma patients. Received: 14 August 1997 / Revised: 6 November 1997 / Accepted: 28 November 1997     

The spectrum of long-term epilepsy-associated tumors: long-term seizure and tumor outcome and neurosurgical aspects

PURPOSE: To describe the histologic spectrum and clinical characteristics of patients with neuroepithelial tumors and drug-resistant epilepsy and to analyze clinical data and treatment related to seizure outcome and survival. METHODS: Data were analyzed from 207 consecutive patients with intractable epilepsy (aged 2-54 years), who between 1988 and 1999 had >or=50% resection of supratentorial, neuroepithelial tumors. Extent of resection was assessed on postoperative magnetic resonance imaging (MRI); seizure outcome was classified according to Engel's outcome scale; and follow-up data were prospectively updated. RESULTS: Median follow-up was eight years (range, 2-14 years). Histologic examination revealed 154 classic epilepsy-associated tumors (ganglioglioma, dysembryoplastic neuroepithelial tumor, pleomorphic xanthoastrocytoma, and pilocytic astrocytomas) and 53 others (astrocytomas and oligodendrogliomas). Four World Health Organization (WHO) grade III tumors were found (astrocytoma, n = 3; ganglioglioma, n = 1). After surgery, 82% of the patients were seizure free (class I). The following factors were associated with improved seizure outcome: Short duration of epilepsy before surgery, single EEG focus, absence of additional hippocampal sclerosis or cortical dysplasia, transsylvian approach, other than astrocytomas, and complete tumor resection. After 5 years, only nine (4%) patients had tumor recurrence, four (2%) with malignant transformation and death. None of the four patients with anaplastic tumors died. Even patients with astrocytomas of WHO grade II or III showed 10-year recurrence of only 25% and 10-year survival of 90%. CONCLUSIONS: Tumors associated with long-term epilepsy should be removed early for two different reasons: high rate of seizure freedom and rare but potential risk of malignant tumor progression. The unexpected long survival of these astrocytomas should be investigated by using immunohistochemistry and molecular biology.     

Overexpression of ADK in human astrocytic tumors and peritumoral tissue is related to tumor-associated epilepsy

Purpose: Adenosine kinase (ADK), a largely astrocyte‐based metabolic enzyme, regulates adenosine homeostasis in the brain. Overexpression of ADK decreases extracellular adenosine and consequently leads to seizures. We hypothesized that dysfunction in the metabolism of tumor astrocytes is related to changes in ADK expression and that those changes might be associated with the development of epilepsy in patients with tumors. Methods: We compared ADK expression and cellular distribution in surgically removed tumor tissue (n = 45) and peritumoral cortex (n = 20) of patients with glial and glioneuronal tumors to normal control tissue obtained at autopsy (n = 11). In addition, we compared ADK expression in tumor patients with and without epilepsy. To investigate ADK expression, we used immunohistochemistry and Western blot analysis. ADK activity measurement was performed in surgical specimens of astrocytomas World Health Organization (WHO) grade III (n = 3), peritumoral cortex (n = 3), and nonepileptic cortex (n = 3). Key Findings: Immunohistochemistry predominantly showed cytoplasmic labeling in tumors and peritumoral tissue containing infiltrating tumor cells. ADK immunoreactivity was significantly stronger in tumor and peritumoral tissue compared to normal white matter and normal cortex, especially in astrocytoma WHO grade III, as confirmed by Western blot analysis and ADK activity measurements. Importantly, we found a significantly higher expression of ADK in the peritumoral infiltrated tissue of patients with epilepsy than in patients without epilepsy. Significance: These results suggest a dysregulation of ADK in astrocytic brain tumors. Moreover, the upregulation of ADK observed in peritumoral infiltrated tissue of glioma patients with epilepsy supports the role of this enzyme in tumor‐associated epilepsy.     

Astrocytic tumors in children: treatment results from a single institution

Objective The aims of this study are to evaluate the patients with astrocytomas and to investigate survival rates and prognosis. Patients and methods Five hundred fourteen patients diagnosed with brain tumor between 1972 and 2003 were retrospectively analyzed. Three different chemotherapy regimens were used according to years. CCNU-based protocols were used in the early years; COPP (cyclophosphamide, oncovin, procarbazine, prednisolone) and CDDP+VP16 (cisplatinum + etoposide) were the other protocols used in the following years. Radiotherapy was used after 3 years of age according to protocols. Results Ninety-eight (19%) out of 514 patients have astrocytic histopathology. The histopathologic distribution was as follows: low grade, 55 patients; high grade, 43 patients. COPP regimen was given to 24 patients, CCNU-based regimen to 13, and CDDP+VP16 to 10 patients. We did not use any chemotherapy in 51 patients. Overall survival (OS) and event free-survival rates were 59.2 and 45.7% in whole group. OS rates were 93.3 and 22.4% for low-grade and high-grade histopathology, respectively (p=0.0001). OS for CCNU, CDDP+VP16, and COPP were 35.9, 22.8, and 30.4%, respectively. Conclusion Low-grade astrocytomas are highly responsive to the surgery, and they do not need any further treatment unless the patient has relapse or recurrence. Still, the treatment of the high-grade tumors is a problem, and it needs new treatment approaches.     

MRI评分在星形细胞起源肿瘤诊断中的应用

目的观察星形细胞起源肿瘤患者的MRI表现,探讨MRI评分与病理学分级之间的相关性。方法选择经手术病理证实的幕上星形细胞起源肿瘤患者44例,其中星形细胞瘤9例、间变性星形细胞瘤14例及多形性胶质母细胞瘤21例,手术后进行MRI评分。评分标准包括肿瘤是否越过中线、有无水肿、不均质性、出血、边界清晰、囊变或坏死、血管流空效应、强化程度和强化的不均质性等9项指标。结果MRI评分随肿瘤病理分级的提高而增加,星形细胞瘤患者的不均质性、水肿、边界清晰、血管流空效应、强化程度和强化的不均质性等6项指标均低于间变性星形细胞瘤和多形性胶质母细胞瘤,其差异具有显著性意义(均P<0.05);而间变性星形细胞瘤与多形性胶质母细胞瘤之间9项指标相比,仅不均质性和强化程度2项差异有显著性意义(P<0.05)。结论MRI评分对选择星形细胞起源肿瘤的治疗方法及判断患者预后具有重要临床参考价值。